The main factors to be considered for successful immunolabeling may be summarized as follows: (1) retention of antigens within the tissue and preservation of their antigenicity; (2) preservation of adequate ultrastructure; and (3) absence of a barrier likely to prevent penetration of the

antibodies into the tissue and interaction with their respective antigens. The achievement of the optimal balance of these three factors is often a rather demanding task. It seems obviously pointless to obtain labeling in the absence of good ultrastructure, and vice versa. Therefore, primary fixation represents a necessary preliminary step to all ICC procedures, although ultrathin frozen sections of unfixed material or cryosubstitution techniques might represent useful alternatives.26,35-38,46,88,112 However, these methods are very expensive, show many technical difficulties, and their general practicability is still limited.